IL-4 and IFN (alpha and gamma) exert opposite regulatory effects on the development of cytolytic potential by Th1 or Th2 human T cell clones.

The cytolytic potential of a total number of 118 CD4+ human T cell clones specific for purified protein derivative (PPD) from Mycobacterium tuberculosis, tetanus toxoid, Lolium perenne group I allergen (Lol p I), Poa pratensis group IX allergen (Poa p IX), or Toxocara canis excretory/secretory antigen(s) (TES) was assessed by both a lectin (PHA)-dependent and a MHC-restricted lytic assay and compared with their profile of cytokine secretion. The majority of clones with Th1 or Th0 cytokine profile exhibited cytolytic activity in both assays, whereas Th2 clones usually did not. There was an association between the cytolytic potential of T cell clones and their ability to produce IFN-gamma, even though IFN-gamma produced by T cell clones was not responsible for their cytolytic activity. IL-4 added in bulk culture before cloning inhibited not only the differentiation of PPD-specific T cells into Th1-like cell lines and clones, but also the development of their cytolytic potential. The depressive effect of IL-4 on the development of PPD-specific T cell lines with both Th1 cytokine profile and cytolytic potential was dependent on early addition of IL-4 in bulk cultures. In contrast, the addition in bulk culture of IFN-gamma enhanced both the cytolytic activity of PPD-specific T cell lines, as well as the proportion of PPD-specific T cell clones with cytolytic activity. The addition in bulk cultures before cloning of IFN-gamma or IFN-alpha favored the development of TES-specific and Poa p IX-specific T cells into T cell clones showing a Th0 or even a Th1, rather than a Th2, cytokine profile. Accordingly, most of TES- and Poa p IX-specific T cell clones derived from cultures containing IFN-gamma or IFN-alpha displayed strong cytolytic activity. These data indicate that the majority of human T cell clones that produce IFN-gamma, but not IL-4 (Th1-like), as well as of T cell clones that produce IFN-gamma in combination with IL-4 (Th0-like) are cytolytic. More importantly, they demonstrate that the addition of IFN (alpha and gamma) or IL-4 in bulk cultures before cloning may influence not only the cytokine profile of human CD4+ T cell clones but also their cytolytic potential.     

Anti-CD3 antibody induces unresponsiveness to IL-2 in Th1 clones but not in Th2 clones

Culture of murine T cells with immobilized (platebound) anti-CD3 antibody results in autocrine growth factor secretion in both Th1 (IL-2 producing) and Th2 (IL-4 producing) cells. Using a panel of murine T cell clones, we demonstrate that the IL-2-induced proliferation of Th1 clones is dramatically inhibited by immobilized anti-CD3 antibody, whereas that of Th2 clones is not. This unresponsiveness of Th1 clones to IL-2 is not due to decreases in IL-2R expression. Supernatants from Th1 or Th2 cell cultures fail to alter the effects of anti-CD3 on the two types of clones, suggesting that unresponsiveness induced in Th1 clones or the lack thereof in Th2 clones is not mediated by a stable cytokine(s). Accessory cells enhance the proliferation of Th1 cells exposed to low concentrations of anti-CD3, but the unresponsiveness induced by high concentrations of anti-CD3 is not prevented by accessory cells. Finally, soluble anti-CD4 antibody prevents the induction of the unresponsive state even at high concentrations of anti-CD3. These experiments demonstrate that two subsets of cloned CD4+ T cells differ in their responses to anti-CD3, and that CD4 molecules may play a critical role in regulating the outcome of receptor-mediated stimulation.     

Role of IFN-gamma in Th1 differentiation: IFN-gamma regulates IL-18R alpha expression by preventing the negative effects of IL-4 and by inducing/maintaining IL-12 receptor beta 2 expression

Two key events occur during the differentiation of IFN-gamma-secreting Th1 cells: up-regulation of IL-12Rbeta2 and IL-12-driven up-regulation of IL-18Ralpha. We previously demonstrated that IL-12-driven up-regulation of IL-18Ralpha expression is severely impaired in IFN-gamma(-/-) mice. However, it was unclear from these studies how IFN-gamma influenced IL-18Ralpha since IFN-gamma alone had no direct effect on IL-18Ralpha expression. In the absence of IL-4, IL-12-dependent up-regulation of IL-18Ralpha/IL-12Rbeta2 was independent of IFN-gamma. However, in the presence of IL-4, IFN-gamma functions to limit the negative effects of IL-4 on both IL-18Ralpha and IL-12Rbeta2. Neutralization of IL-4 restored IL-12-driven up-regulation of IL-18Ralpha/IL-12Rbeta2 in an IFN-gamma-independent fashion. In the absence of both IL-12 and IL-4, IFN-gamma up-regulates IL-12beta2 expression and primes IFN-gamma-producing Th1 cells. When T cells were primed in the presence of IL-4, no correlation was found between the levels of expression of the IL-18Ralpha or the IL-12Rbeta2 and the capacity of these cells to produce IFN-gamma, suggesting that IL-4 may also negatively affect IL-12-mediated signal transduction and thus Th1 differentiation. These data clarify the role of IFN-gamma in regulation of IL-18Ralpha/IL-12Rbeta2 during both IL-12-dependent and IL-12-independent Th1 differentiation.     

IL-4 plays a dominant role in the differential development of Tho into Th1 and Th2 cells.

We have analyzed the evolution of the pattern of lymphokine secretion by Th cell lines specific for either the synthetic terpolymer Glu60Ala30Tyr10 (GAT) or killed bacillus Calmette Guérin. When cultured in the presence of exogenous rIL-2 as a growth factor, GAT-specific Th cell lines secreted mainly IL-4, whereas bacillus Calmette Guérin-specific lines produced predominantly IL-2. However, culturing in the presence of rIL-4 or of anti-IL-4 mAb and rIL-2 led to the establishment of Th2-like and Th1-like lines, respectively, regardless of their Ag specificity. Inasmuch as we show that the proliferative response of mature Th1 and Th2 cells was identical in the presence of IL-4, these results indicate that IL-4 influences the development of Th cell subsets. To understand the mode of IL-4 action, we isolated immature GAT-specific Tho clones able to secrete IL-2 and IL-4. Two types of Tho cells were isolated: ThoA cells that secreted IL-2 and IL-4, but not IFN-gamma, and ThoB cells that secreted IL-2, IL-4, and IFN-gamma. We show for the first time that such cells are indeed Th precursors able to differentiate into Th1 or Th2 cells. We demonstrate that IL-4 positively and negatively controls the differentiation of Tho cells into Th2 and Th1 cells, respectively. When cultured in rIL-4, Tho cells stop secreting IL-2 and IFN-gamma, but maintain IL-4 secretion. Moreover, endogenous IL-4 produced by Tho cells has similar effects: when cultured in rIL-2 alone, Tho cells either keep their immature phenotype or become Th2 cells, but do not become Th1 cells. In contrast, neutralization of secreted IL-4 completely prevents the differentiation of Tho into Th2 cells, but permits the development of Th1 cells. The presence of exogenous IFN-gamma does not affect the development of Tho into Th1 and Th2 cells, because it does not modify either mode of IL-4 action. However, it influences the ratio between the two types of Tho cells: when IL-4 is neutralized, added IFN-gamma can induce IFN-gamma secretion by ThoA cells and thereby facilitate their passage into ThoB cells. Taken together, our results demonstrate that IL-4, in addition to mediating T cell growth, is a principal factor that controls the differential development of Tho cells into Th1 and Th2 cells.     

Simultaneous production of IL-2, IL-4, and IFN-gamma by activated human CD4+ and CD8+ T cell clones

In the present study, we have investigated the ability of human T cells to secrete IL-2, IL-4, and IFN-gamma. IL-4 and IFN-gamma were quantified with enzymatic immunoassays and IL-2 with a biologic assay by using the murine IL-2-dependent cell line CTLL-2. PBL, stimulated with Con A or with a combination of the phorbol ester 13-O-tetradecanoylphorbol-12-acetate and the Ca2+ ionophore A23187 secreted IL-2, IL-4, and IFN-gamma. The kinetics of the secretion of the three lymphokines was investigated with two CD4+ clones; one (GEO-2) that produced IL-2, IL-4, and IFN-gamma and another (HY640), that produced only IL-2 and IFN-gamma. Significant IL-2, IL-4, and IFN-gamma production was observed after only 8 h of activation. Maximal levels of IL-2 and IL-4 were found 20 h after the onset of the stimulation which subsequently decreased. In contrast, IFN-gamma levels continued to increase in a period up to 40 h and then leveled off. In spite of these differences in secretion, the kinetics of accumulation of mRNA did not differ. The IL-2, IL-4, and IFN-gamma mRNA were detectable 2 h after stimulation and continued to accumulate for a period up to 20 h. In a series of 22 CD4+ clones, 21 were able to secrete all three lymphokines upon stimulation. Almost all CD8+ clones were able to produce IL-2 and IFN-gamma, but only six of the 23 CD8+ T cell clones secreted IL-4. In addition, five CD4+ (allo)antigen-specific T cell clones were tested for IL-2, IL-4, and IFN-gamma secretion upon specific stimulation. Two alloantigen-specific and two tetanus toxoid-specific T cell clones secreted IL-2, IL-4, and IFN-gamma simultaneously, whereas one alloantigen-specific T cell clone secreted IL-2 and IFN-gamma, but not IL-4. A supernatant of the CD4+ T cell clone GEO-2, that contained high levels of IFN-gamma and IL-4, was unable to induce the low affinity receptor for IgE, CD23, on a Burkitt lymphoma cell line. However, after separation of IL-4 from IFN-gamma by using HPLC, the IL-4-containing fraction-induced CD23, which could be blocked by the fraction that contained IFN-gamma and by a polyclonal rabbit anti-IL-4 antiserum. Finally, the partly purified IL-4, that was devoid of IL-2, promoted the growth of the clone GEO-2.     

IFN-gamma modulates the early development of Th1 and Th2 responses in a murine model of cutaneous leishmaniasis.

Resistance to Leishmania major in mice is associated with the generation of distinct CD4+ Th subsets, termed TH1 and TH2. To define the factors contributing to the genesis of these Th cells, we first investigated when these subsets developed following L. major infection. Lymph node (LN) cells collected 3 days after infection of BALB/c mice secreted IL-4 and IL-5 in vitro, but little IFN-gamma, whereas LN cells from a resistant strain, C3H/HeN, secreted IFN-gamma and no IL-4 or IL-5. Cytokine production was eliminated in both cases by in vivo or in vitro depletion of CD4+ cells, but not after depletion of CD8+ cells. Similar responses were observed after inoculation of killed promastigotes or a soluble leishmanial Ag preparation. These data indicate that the development of Th1- and Th2-like responses can precede lesion formation and does not require a live infection. We next investigated whether IFN-gamma was important in the differentiation of Th1 and Th2 cells. C3H/HeN mice have previously been shown to be susceptible to leishmanial infection after treatment with anti-IFN-gamma. We confirmed this observation and found that the abrogation of resistance was associated with enhanced production of IL-4 and IL-5, and decreased production of IFN-gamma by cells taken from these mice. Conversely, LN cells from BALB/c mice inoculated with parasites plus IFN-gamma produced significantly higher levels of IFN-gamma, and decreased levels of IL-4 and IL-5, than mice infected with parasites alone. Finally, we determined if IFN-gamma might augment vaccine induced immunity. We found that s.c. immunization with soluble leishmanial Ag, the bacterial adjuvant, Corynebacterium parvum and IFN-gamma could protect mice against L. major infection, and that this protection was associated with induction of Th1 responses. From these data we conclude that levels of IFN-gamma at the time of infection or immunization dramatically alters the type of response elicited: high levels of IFN-gamma favor Th1 type responses, whereas low levels promote a Th2 response.     

Ribavirin exerts differential effects on functions of cd4(+) Th1, th2, and regulatory T cell clones in hepatitis C

Ribavirin improves outcomes of therapy in chronic hepatitis C but its mode of action has still remained unclear. Since ribavirin has been proposed to modulate the host's T cell responses, we studied its direct effects on CD4(+) T cell clones with diverse functional polarization which had been generated from patients with chronic hepatitis C. We analysed in vitro proliferation ([(3)H] thymidine uptake) and cytokine responses (IL-10, IFN-gamma) at varying concentrations of ribavirin (0-10μg/ml) in 8, 9 and 7 CD4(+) TH1, TH2 and regulatory T cell (Treg) clones, respectively. In co-culture experiments, we further determined effects of ribarivin on inhibition of TH1 and TH2 effector cells by Treg clones. All clones had been generated from peripheral blood of patients with chronic hepatitis C in the presence of HCV core protein. Ribavirin enhanced proliferation of T effector cells and increased production of IFN-gamma in TH1 clones, but had only little effect on IL-10 secretion in TH2 clones. However, ribavirin markedly inhibited IL-10 release in Treg clones in a dose dependent fashion. These Treg clones suppressed proliferation of T effector clones by their IL-10 secretion, and in co-culture assays ribavirin reversed Treg-mediated suppression of T effector cells. Our in vitro data suggest that - in addition to its immunostimulatory effects on TH1 cells - ribavirin can inhibit functions of HCV-specific Tregs and thus reverses Treg-mediated suppression of T effector cells in chronic hepatitis C.     

Shift in Th1 (IL-2 and IFN-gamma) and Th2 (IL-10 and IL-4) cytokine mRNA balance within two new histological main-types of rheumatoid-arthritis (RA).

Cytokines are the main mediators of inflammation in rheumatoid arthritis (RA). Thus, Th2 cytokines--such as IL-4 and IL-10--have protective properties to this disease. In opposite, the Th1 cytokines--such as IL-2 and IFN-gamma--are supporting proinflammatory microenvironment in joints from patients with RA. The imbalance of Th1/Th2 cytokine steady state may play an important role in the pathogenesis of rheumatoid arthritis. The evaluation of this imbalance leads up to the possibility of pathohistological discrimination in this disease. In this context, we investigated Th1- (IFN-gamma, IL-2) and Th2 (IL-10, IL-4)-cell-derived cytokine mRNA expression in two novel pathohistological main-types of RA synovial membrane (SM). These main-types are characterized by different tissue-infiltrating inflammatory cells and different extent of SM destruction. Our findings showed that expression of IL-10 mRNA was an outcome of histological main-type I (p<0.001), whereas expression of IFN-gamma and IL-2 were mainly associated with pathohistological main-type II (p<0.005, p<0.05). Surprisingly, IL4 was not differential expressed and could be associated with another special T cell subset in this disease. These results suggest that Th1/Th2 balance is biased to Th2 cytokines within main-type I and Th1 cytokines in main-type II.     

IL-4 directs the development of Th2-like helper effectors

Our studies show that the presence of IL-4 during the response of naive Th cells causes precursors to develop into a population comprised largely of "Th2-like" effectors that secrete IL-4 and IL-5, but little IL-2 or IFN-gamma. We find that the levels of IL-4 and IL-2 determine both the level of effectors developed in response to mitogen or Ag and the patterns of lymphokines they secrete when restimulated. IL-2 is required for optimum generation of effectors, and increasing levels of IL-2, augments the expansion of effectors secreting both IL-4/IL-5 and IFN-gamma. In contrast, IL-4 is required for the development of IL-4/IL-5 secreting effectors but suppresses the development of IL-2 and at higher doses IFN-gamma-secreting effectors detected after 4 days. Also dramatic are the effects of the presence or absence of IL-4 evaluated after an additional 1 to 2 wk. When cultures with or without initial IL-4 are cultured in IL-2 alone from days 4 to 11, they retain their distinct patterns of lymphokine production. Those cells that developed in cultures without IL-4 progressively secrete more IL-2 and can be maintained and expanded in IL-2. They continue to produce IFN-gamma, though the levels decrease somewhat with time, but they do not acquire the ability to produce IL-4 or IL-5. These cells thus increasingly resemble Th1 cell lines. In contrast, those cells in cultures initially exposed to IL-4, generate effectors which secrete high levels of IL-4/IL-5 (plus variable levels of IFN-gamma) at days 4 to 5, but the populations of cells developed, are not maintained well on IL-2 alone. Those cells that do survive continue to secrete IL-4 and IL-5 but not IL-2. In addition, IFN-gamma production, if present, falls off with time. Thus the cells in these cultures take on an increasingly Th2-like phenotype. It appears that the effects of low levels of IL-4 in suppressing IL-2 production by day 4 effectors appear to be transient whereas the higher levels appear to drive the development along a distinct pathway which is irreversible. These studies support the concept that different subsets of helper cells, which correspond roughly to Th1 and Th2 subsets, can develop rapidly in short term culture with respectively low vs high levels of IL-4. They support the concept that such distinct phenotypes arise from alternate pathways of differentiation that can be expected to reflect pathways available for helper T cell differentiation in the animal.     

Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.     

Antibodies to the IL-12 receptor beta 2 chain mark human Th1 but not Th2 cells in vitro and in vivo

Great attention has been placed on the possibility of distinguishing Th1 from Th2 cells on the basis of differential expression of surface receptors. We have recently shown that the differential expression of the IL-12R beta 2 chain in Th1 and Th2 cells, as measured at the mRNA level, accounts for an important regulatory mechanism in the differentiation of the two cell subsets. In this study, we identify IL-12R expression at the protein level. We have generated an anti-IL-12R beta 2-specific mAb and analyzed IL-12R beta 2 expression on polarized Th cell populations generated in vitro and on T cells derived from patients with Th1- or Th2-mediated inflammatory conditions. Although IL-12R beta 2 was absent in freshly isolated PBMC and in cord blood cells, we were able to detect IL-12R beta 2 expression selectively in differentiated Th1 and T cytotoxic 1, but not Th2 or T cytotoxic 2 cells. In the presence of IL-12, cell surface expression of the IL-12R beta 2 subunit was readily detected on T cells after 24 h, reached the maximum at day 5, and declined thereafter. Most importantly, the anti-IL-12R beta 2 mAb recognizes lung T cells from patients with sarcoidosis, a disease characterized by a typical cell-mediated, Th1-type inflammatory response. In contrast, IL-12R beta 2 was absent in lung T cells from patients with allergic asthma, a disease characterized by a Th2-type inflammatory response. The mAb reported in this study should represent a powerful tool to investigate the role of Th1 and Th2 cells in inflammatory conditions and to monitor therapies aimed at altering the balance of Th cell subsets.     

Reciprocal regulation of permeability through a cultured keratinocyte sheet by IFN-gamma and IL-4

The T cell cytokines profoundly modify the phenotypic and functional characteristics of keratinocytes. Until now, no study has focused on the effect of Th1 and Th2 cytokines on keratinocyte permeability. Using a two-layer well culturing system, permeability was assessed through cultured keratinocyte sheet in the presence or absence of various concentrations of IFN-gamma and IL-4. Transepithelial electrical resistance (TER) and the flux of 40 kDa FITC-dextrans were measured across the cultured keratinocyte sheet. IFN-gamma significantly increased the TER in a dose- and time-dependent manner, suggesting that IFN-gamma profoundly inhibited the permeability of ions through the keratinocyte sheet. In contrast, IL-4 did not affect the TER. When compared to medium control, the flux of FITC-dextran of the IFN-gamma group was significantly decreased in a dose-dependent fashion. In sharp contrast, the flux of FITC-dextran was significantly and dose-dependently increased in the presence of IL-4. A significant increase in TER and a significant decrease in the flux of dextran suggested that IFN-gamma clearly reduced the permeability of both ions and high molecular weight material through the keratinocyte sheet. Although IL-4 did not affect the permeability of the ions, it significantly enhanced the permeability of high molecular weight material. A flow cytometric assay revealed that the expression of desmoglein-3 was suppressed by IL-4, but was enhanced by IFN-gamma. The reciprocal regulation of permeability of the cultured keratinocyte sheet by IFN-gamma and IL-4 may be partly related to the modification of intercellular adhesion molecules.     

IL-2, IL-6, and IFN-gamma have distinct effects on the IL-4 plus PMA-induced proliferation of thymocyte subpopulations

We have previously reported complex effects of cytokine-containing T cell supernatants on the interleukin (IL)4 plus phorbol 12-myristate 13-acetate (PMA)-induced proliferative response of murine thymocytes. Here we show that recombinant murine IL-2, IL-6, and IFN-gamma each differentially regulate the IL-4/PMA-driven growth of thymocyte subpopulations. Thymocytes fractionated into four subpopulations on the basis of CD4 and CD8 expression were stimulated to proliferate by IL-4/PMA. Interferon-gamma (IFN-gamma) caused almost complete inhibition of the CD4+/CD8- response but had no measurable effect on the growth of CD4-/CD8+ or CD4-/CD8- populations. This inhibitory effect was also observed on splenic CD4+/CD8- T cells. In contrast, IL-6 strongly enhanced the proliferative response of CD4+/CD8- thymocytes, but showed no effect on peripheral CD4+/CD8- T cells, suggesting that IL-6 may be an important regulator of growth in the thymus. IL-2 also enhanced the proliferation of both CD4-/CD8+ and CD4-/CD8- thymocytes to IL-4 and PMA. To test whether the IL-4/PMA stimulus provided all the signals required to initiate growth in each subpopulation, we titrated cell number and examined the relationship between cell dose and cell response. Growth of CD8+/CD4- cells was cell density independent, indicating that IL-4/PMA is sufficient stimulus to induce growth of these cells. In contrast, growth of CD4-/CD8- and CD4+/CD8- cells is cell density dependent, suggesting a requirement for another signal provided by the cells themselves. These observations suggest that more signals remain to be identified in this thymocyte growth system.     

The cytotoxic process of CD4 Th1 clones.

Previous studies have demonstrated that murine CD4 Th1 cells lack perforin and use a pathway distinctive from CD8 CTL to express cytotoxicity. Whether the cytotoxic process of Th1 cells can be separated into identifiable stages and how these differences affect this process were determined in this study. We have resolved the cytotoxic process of Th1 clones into three stages identical with those of CD8 CTL, namely, conjugate formation/activation, lethal hit, and effector-independent programming for target DNA fragmentation. By comparing the cytotoxic processes between Th1 clones on Ag-pulsed targets and (PMA+A23187)-activated Th1 clones on unpulsed targets, we have also demonstrated that 1) the requirement of CD4 Th1 cells for de novo synthesis of cytotoxic machinery was partly responsible for the lag time in the induction of target DNA fragmentation by Th1 clones; 2) lethal hit was delivered rapidly; 3) lethal hit under forced contact by centrifugation did not need extracellular Ca2+ and Mg2+; 4) without centrifugation, lethal hit required extracellular Mg2+, but not Ca2+; 5) the average functional half life of the cytotoxic machinery was 54 +/- 24 (n = 4) min. The data demonstrate that the cytotoxic process of Th1 clones uses an activation-dependent cytotoxic machinery to deliver a short-lived, short-ranged, and quick-acting lethal hit to target, which induces a program in target for DNA fragmentation.     

Natural human IL-1 beta exhibits regulatory actions on human bone-derived cells in vitro

We have shown that natural homogenous IL-1 beta exhibits regulatory activities on human bone-derived osteoblast-like cells in vitro. IL-1 beta stimulated cellular proliferation and the synthesis of prostaglandin E2 and plasminogen activator activity by the cultured human osteoblast-like cells. In contrast to these stimulatory actions, IL-1 beta antagonised the stimulatory effects of 1.25(OH)2 D3 on the production of alkaline phosphatase and osteocalcin, two markers of the osteoblast phenotype. These studies indicate that this cytokine may therefore have potential physiological and pathological effects on bone metabolism.     

Antiproliferative effect of IFN-gamma in immune regulation. III. Differential selection of TH1 and TH2 murine helper T lymphocyte clones using recombinant IL-2 and recombinant IFN-gamma

Supernatants collected after primary or secondary stimulation of spleen cells contain different arrays of lymphokines. Primary supernatants from spleen cells stimulated with Con A or allogeneic spleen cells (MLC-SF) contain IL-2 but little IL-4 or IGN-gamma; in contrast, secondary MLC-SF contains IL-2 as well as substantial IL-4 and IFN-gamma. Our laboratory previously had always used secondary MLC-SF for cloning T cells, and had routinely obtained TH1 helper T lymphocyte clones. In the present study, when primary Con A-SF was used as source of growth factors, TH2 and not TH1 clones were preferentially derived. Considering the possibility that IFN-gamma may be one important factor in determining whether TH1 or TH2 clones are preferentially obtained, clone derivation was then performed either in the presence of rIL-2 or rIL-2 plus rIFN-gamma. The majority of clones derived using rIL-2 alone were TH2 cells, whereas the majority of clones derived using rIL-2 plus rIFN-gamma were TH1 cells. Using either procedure, some clones were obtained that produced IL-2, IL-4, and IFN-gamma. These data are consistent with our previous observations that IFN-gamma inhibits the proliferation of TH2 but not TH1 clones, and suggest that the presence of IFN-gamma during an immune response would result in the preferential expansion of helper T lymphocytes of the TH1 phenotype. Our procedure for the differential selection of TH1 and TH2 clones reactive with the same Ag should be useful for designing in vitro systems for studying the function of these cell subsets in specific immune responses.     

Comparison of lymphokine secretion and responsiveness of human T cell clones isolated in IL-4 and in IL-2.

Interleukin (IL)-4 has been shown to be secreted simultaneously with IL-2 and interferon (IFN)-gamma by the majority of CD4+ human T cell clones isolated and cultured using IL-2 as a growth factor. Moreover, IL-4 was found to be as efficient as IL-2 to promote the outgrowth of human T cell clones. In this study we have investigated the pattern of lymphokine production by human T cell clones isolated and cultured in IL-4. Most of the CD4+ T cell clones isolated in IL-4 were found to have the ability to simultaneously secrete IL-2, IL-4, and IFN-gamma upon activation. The T cell clones isolated in IL-4 produced, in general, more IL-4 and less IL-2 than the clones isolated and cultured in IL-2. This tendency did not appear to be a stable feature inasmuch as when representative CD4+ T cell clones were split and cultured in either IL-2 or IL-4, the clones in IL-2 secreted more IL-2 and less IL-4 than the same cells cultured in IL-4. These results indicate that the isolation and culture of human CD4+ T cells in IL-4 did not lead to an "irreversible" development of these cells into Th-1- or Th-2-like cells. Clones isolated in IL-4 responded better to IL-4 than they did to IL-2. On the other hand, T cell clones from the same donor isolated in IL-2 showed the reverse pattern since these latter cells were found to respond better to IL-2 than to IL-4. Furthermore, "nonresponsiveness" of a T cell clones in a [3H]TdR assay to either IL-2 or IL-4 is not a stable feature since clones, unresponsive to a particular lymphokine, could be adapted to become responsive.